Biomedical subjects

C Robert Matthews

Publications and source records attributed to C Robert Matthews.

22 records · Page 2Linked to original sources

PubMed2002

Folding mechanism of indole-3-glycerol phosphate synthase from Sulfolobus solfataricus: a test of the conservation of folding mechanisms hypothesis in (beta(alpha))(8) barrels.

As a test of the hypothesis that folding mechanisms are better conserved than sequences in TIM barrels, the equilibrium and kinetic folding mechanisms of indole-3-glycerol phosphate synthase (sIGPS) from the thermoacidophilic archaebacterium Sulfolobus solfataricus were compared to the well-characterized models of the alpha subunit of tryptophan synthase (alphaTS) from Escherichia coli. A multifaceted approach combining urea denaturation and far-UV circular dichroism, tyrosine fluorescence total intensity, and tyrosine fluorescence anisotropy was employed. Despite a sequence identity of only 13%, a stable intermediate (I) in sIGPS was found to be similar to a stable intermediate in alphaTS in terms of its thermodynamic properties and secondary structure. Kinetic experiments revealed that the fastest detectable folding event for sIGPS involves a burst-phase (<5ms) reaction that leads directly to the stable intermediate. The slower of two subsequent phases reflects the formation/disruption of an off-pathway dimeric form of I. The faster phase reflects the conversion of I to the native state and is limited by folding under marginally stable conditions and by isomerization or rearrangement under strongly folding conditions. By contrast, alphaTS is thought to fold via an off-pathway burst-phase intermediate whose unfolding controls access to a set of four on-pathway intermediates that comprise the stable equilibrium intermediate. At least three proline isomerization reactions are known to limit their interconversions and lead to a parallel channel mechanism. The simple sequential mechanism deduced for sIGPS reflects the dominance of the on-pathway burst-phase intermediate and the absence of prolyl residues that partition the stable intermediate into kinetically distinguishable species. Comparison of the results for sIGPS and alphaTS demonstrates that the thermodynamic properties and the final steps of the folding reaction are better conserved than the early events. The initial events in folding appear to be more sensitive to the sequence differences between the two TIM barrel proteins.

Amino Acid Sequence↗

PubMed2002

Highly divergent dihydrofolate reductases conserve complex folding mechanisms.

To test the hypothesis that protein folding mechanisms are better conserved than amino acid sequences, the mechanisms for dihydrofolate reductases (DHFR) from human (hs), Escherichia coli (ec) and Lactobacillus casei (lc) were elucidated and compared using intrinsic Trp fluorescence and fluorescence-detected 8-anilino-1-naphthalenesulfonate (ANS) binding. The development of the native state was monitored using either methotrexate (absorbance at 380 nm) or NADPH (extrinsic fluorescence) binding. All three homologs displayed complex unfolding and refolding kinetic mechanisms that involved partially folded states and multiple energy barriers. Although the pairwise sequence identities are less than 30 %, folding to the native state occurs via parallel folding channels and involves two types of on-pathway kinetic intermediates for all three homologs. The first ensemble of kinetic intermediates, detected within a few milliseconds, has significant secondary structure and exposed hydrophobic cores. The second ensemble is obligatory and has native-like side-chain packing in a hydrophobic core; however, these intermediates are unable to bind active-site ligands. The formation of the ensemble of native states occurs via three channels for hsDHFR, and four channels for lcDHFR and ecDHFR. The binding of active-site ligands (methotrexate and NADPH) accompanies the rate-limiting formation of the native ensemble. The conservation of the fast, intermediate and slow-folding events for this complex alpha/beta motif provides convincing evidence for the hypothesis that evolutionarily related proteins achieve the same fold via similar pathways.

Amino Acid Sequence↗

PubMed2002

Analysis of kinetics using a hybrid maximum-entropy/nonlinear-least-squares method: application to protein folding.

A hybrid analysis that combines the maximum entropy method (MEM) with nonlinear least squares (NLS) fitting has been developed to interpret a general time-dependent signal. Data that include processes of opposite sign and a slow baseline drift can be inverted to obtain both a continuous distribution of lifetimes and a sum of discrete exponentials. Fits by discrete exponentials are performed with initial parameters determined from the distribution of lifetimes obtained with the MEM. The regularization of the parameter space achieved by the MEM stabilizes the introduction of each successive exponential in the NLS fits. This hybrid approach is particularly useful when fitting by a large number of exponentials. Revision of the MEM "prior" based on features in the data can improve the lifetime distribution obtained. Standard errors in the mean are estimated automatically for raw data. The results presented for simulated data and for fluorescence measurements of protein folding illustrate the utility and accuracy of the hybrid algorithm. Analysis of the folding of dihydrofolate reductase reveals six kinetic processes, one more than previously reported.

Algorithms↗

PubMed2000

Localized, stereochemically sensitive hydrophobic packing in an early folding intermediate of dihydrofolate reductase from Escherichia coli.

Mutational analysis was performed to probe the development of hydrophobic clusters during the early events in the folding of dihydrofolate reductase. Replacements were made in several hydrophobic subdomains to examine the roles of hydrophobicity and stereochemistry in the formation of kinetic intermediates. Amide protons in two of these clusters, including residues I91, I94, and I155, have been shown to be protected against solvent exchange within 13 ms of folding. Additional hydrophobic clusters were probed by substitutions at residues I2, I61, and L112; these residues are not protected from exchange until later in the folding reaction. Valine and leucine replacements at positions I91, I94, and I155 significantly diminish the formation of the burst phase kinetic intermediate, relative to the wild-type protein. In contrast, I2 and I61 are insensitive to these substitutions in the first 5 ms of the folding reaction, as is the replacement of L112 with either isoleucine or valine. These results demonstrate that the tightly packed core of dihydrofolate reductase is acquired in a non-uniform fashion, beginning in the submillisecond time frame. The progressive development of specific side-chain packing in localized hydrophobic clusters may be a common theme for complex protein folding reactions.

Amino Acid Substitution↗